Potato is one of the main food crops in China, and research on potato molecular breeding research is of great significance. In the diploid potato, QTL mapping and cloning of important genes has been reported extensively; The development of tetraploid linked analysis software, genetic map construction in tetraploid potato and QTL mapping have also made breakthrough progress in recent years. Meanwhile, molecular markers are an important supplement method for potato breeding and it can quickly and accurately screen out multiple good traits. In this paper, the progress of the QTL mapping, cloning of important agronomic traits in potato and 3 resistance traits linkage markers breeding were summarized, to provid a reference and practical basis for accelerating potato molecular breeding.

Landrace potato cultivars are native to two areas in South America: the high Andes from eastern Venezuela to northern Argentina and the lowlands of south-central Chile. Potato first appeared outside of South America in Europe in 1567 and rapidly diffused worldwide. Two competing hypotheses suggested the origin of the "European" potato from the Andes or from lowland Chile, but the Andean origin has been widely accepted over the last 60 years. All modern potato cultivars predominantly have Chilean germplasm, explained as originating from breeding with Chilean landraces subsequent to the late blight epidemics beginning in 1845 in the UK. The Andean origin has been questioned recently through examination of landraces in India and the Canary Islands, but this evidence is inferential. Through a plastid DNA deletion marker from historical herbarium specimens, we report that the Andean potato predominated in the 1700s, but the Chilean potato was introduced into Europe as early as 1811 and became predominant long before the late blight epidemics in the UK. Our results provide the first direct evidence of these events and change the history of introduction of the European potato. They shed new light on the value of past breeding efforts to recreate the European potato from Andean forms and highlight the value of herbarium specimens in investigating origins of crop plants.

Abstract Potato (Solanum tuberosum L.) is the world's most important non-grain food crop and is central to global food security. It is clonally propagated, highly heterozygous, autotetraploid, and suffers acute inbreeding depression. Here we use a homozygous doubled-monoploid potato clone to sequence and assemble 86% of the 844-megabase genome. We predict 39,031 protein-coding genes and present evidence for at least two genome duplication events indicative of a palaeopolyploid origin. As the first genome sequence of an asterid, the potato genome reveals 2,642 genes specific to this large angiosperm clade. We also sequenced a heterozygous diploid clone and show that gene presence/absence variants and other potentially deleterious mutations occur frequently and are a likely cause of inbreeding depression. Gene family expansion, tissue-specific expression and recruitment of genes to new pathways contributed to the evolution of tuber development. The potato genome sequence provides a platform for genetic improvement of this vital crop. 2011 Macmillan Publishers Limited. All rights reserved

[5]

Meyer RC, MilbourneD, Hackett CA, et al.

Linkage analysis in tetraploid potato and association of markers with quantitative resistance to late blight (Phytophthora infestans)

We have constructed a partial linkage map in tetraploid potato which integrates simplex, duplex and double-simplex AFLP markers. The map consists of 231 maternal and 106 paternal markers with total map lengths of 990.965cM and 484.665cM. The longer of the two cumulative map lengths represents approximately 25% coverage of the genome. In tetraploids, much of the polymorphism between parental clones is masked by `dosage' which significantly reduces the number of individual markers that can be scored in a population. Consequently, the major advantage of using AFLPs – their high multiplex ratio – is reduced to the point where the use of alternative multi-allelic marker types would be significantly more efficient. The segregation data and map information have been used in a QTL analysis of late blight resistance, and a multi-allelic locus at the proximal end of chromosome VIII has been identified which contributes significantly to the expression of resistance. No late blight resistance genes or QTLs have previously been mapped to this location.

This article presents methodology for the construction of a linkage map in an autotetraploid species, using either codominant or dominant molecular markers scored on two parents and their full-sib progeny. The steps of the analysis are as follows: identification of parental genotypes from the parental and offspring phenotypes; testing for independent segregation of markers; partition of markers into linkage groups using cluster analysis; maximum-likelihood estimation of the phase, recombination frequency, and LOD score for all pairs of markers in the same linkage group using the EM algorithm; ordering the markers and estimating distances between them; and reconstructing their linkage phases. The information from different marker configurations about the recombination frequency is examined and found to vary considerably, depending on the number of different alleles, the number of alleles shared by the parents, and the phase of the markers. The methods are applied to a simulated data set and to a small set of SSR and AFLP markers scored in a full-sib population of tetraploid potato.

TetraploidMap is a suite of Fortran 90 routines run from Microsoft Windows with a text-based input and output. TetraploidMap enables the user to assemble a linkage map from dominant and codominant (multiallelic) marker loci scored for the parents and full-sib progeny of a cross in an autotetraploid species. It includes routines for the inference of the parental genotypes, identification of linkage groups, two-point analysis to estimate the recombination frequency and LOD score between all pairs of marker in a linkage group, and locus ordering by simulated annealing.

[9]

Bourke PM, Voorrips RE, Visser RG, et al.

The double-reduction landscape in tetraploid potato as revealed by a high-density linkage map

The creation of genetic linkage maps in polyploid species has been a long-standing problem for which various approaches have been proposed. In the case of autopolyploids, a commonly used simplification is that random bivalents form during meiosis. This leads to relatively straightforward estimation of recombination frequencies using maximum likelihood, from which a genetic map can be derived. However, autopolyploids such as tetraploid potato (Solanum tuberosum L.) may exhibit additional features, such as double reduction, not normally encountered in diploid or allopolyploid species. In this study, we produced a high-density linkage map of tetraploid potato and used it to identify regions of double reduction in a biparental mapping population. The frequency of multivalents required to produce this degree of double reduction was determined through simulation. We also determined the effect that multivalents or preferential pairing between homologous chromosomes has on linkage mapping. Low levels of multivalents or preferential pairing do not adversely affect map construction when highly informative marker types and phases are used. We reveal the double-reduction landscape in tetraploid potato, clearly showing that this phenomenon increases with distance from the centromeres.

[10]

Hackett CA, MilneI, Bradshaw JE, et al.

Tetraploid Map for Windows:linkage map construction and QTL mapping in autotetraploid species

An earlier program, TetraploidMap, enabled linkage analysis to be performed for autotetraploid species, with a text-based input and output. The current program, TetraploidMap for Windows, is considerably enhanced, and now beyond linkage analysis to perform quantitative trait locus (QTL) interval mapping, with a range of models and thresholds assessed by permutation tests. A Windows-based interface facilitates data entry and exploration. TetraploidMap for Windows is freely available from the Web site of Bioinformatics and Statistics Scotland at http://www.bioss.ac.uk/ (user-friendly software).

The objective of this study was to construct a single nucleotide polymorphism (SNP)-based genetic map at the cultivated tetraploid level to locate quantitative trait loci (QTL) contributing to economically important traits in potato (Solanum tuberosumL.). The 156 F1progeny and parents of a cross (MSL603) between “Jacqueline Lee” and “MSG227-2” were genotyped using the Infinium 8303 Potato Array. Furthermore, the progeny and parents were evaluated for foliar late blight reaction to isolates of the US-8 genotype ofPhytophthora infestans(Mont.) de Bary and vine maturity. Linkage analyses and QTL mapping were performed using a novel approach that incorporates allele dosage information. The resulting genetic maps contained 1972 SNP markers with an average density of 1.36 marker per cM. QTL mapping identified the major source of late blight resistance in “Jacqueline Lee.” The best SNP marker mapped 650.54 Mb from a resistance hotspot on the long arm of chromosome 9. For vine maturity, the major-effect QTL was located on chromosome 5 with allelic effects from both parents. A candidate SNP marker for this trait mapped 650.25 Mb from theStCDF1gene, which is a candidate gene for the maturity trait. The identification of markers forP. infestansresistance will enable the introgression of multiple sources of resistance through marker-assisted selection. Moreover, the discovery of a QTL for late blight resistance not linked to the QTL for vine maturity provides the opportunity to use marker-assisted selection for resistance independent of the selection for vine maturity classifications.

To facilitate genome-guided breeding in potato, we developed an 8303 Single Nucleotide Polymorphism (SNP) marker array using potato genome and transcriptome resources. To validate the Infinium 8303 Potato Array, we developed linkage maps from two diploid populations (DRH and D84) and compared these maps with the assembled potato genome sequence. Both populations used the doubled monoploid reference genotype DM1-3 516 R44 as the female parent but had different heterozygous diploid male parents (RH89-039-16 and 84SD22). Over 4,400 markers were mapped (1,960 in DRH and 2,454 in D84, 787 in common) resulting in map sizes of 965 (DRH) and 792 (D84) cM, covering 87% (DRH) and 88% (D84) of genome sequence length. Of the mapped markers, 33.5% were in candidate genes selected for the array, 4.5% were markers from existing genetic maps, and 61% were selected based on distribution across the genome. Markers with distorted segregation ratios occurred in blocks in both linkage maps, accounting for 4% (DRH) and 9% (D84) of mapped markers. Markers with distorted segregation ratios were unique to each population with blocks on chromosomes 9 and 12 in DRH and 3, 4, 6 and 8 in D84. Chromosome assignment of markers based on linkage mapping differed from sequence alignment with the Potato Genome Sequencing Consortium (PGSC) pseudomolecules for 1% of the mapped markers with some disconcordant markers attributable to paralogs. In total, 126 (DRH) and 226 (D84) mapped markers were not anchored to the pseudomolecules and provide new scaffold anchoring data to improve the potato genome assembly. The high degree of concordance between the linkage maps and the pseudomolecules demonstrates both the quality of the potato genome sequence and the functionality of the Infinium 8303 Potato Array. The broad genome coverage of the Infinium 8303 Potato Array compared to other marker sets will enable numerous downstream applications.

[16]

Bonierbale MW, Plaisted RL, Tanksley SD.

RFLP maps based on a common set of clones reveal modes of chromosomal evolution in potato and tomato

Abstract Potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum) are members of the Solanaceae (nightshade family) and have the same basic chromosome number (x = 12). However, they cannot be cross-hybridized and, until now, it was unknown how conserved the gene order might be between these two species. We report herein the construction of a genetic linkage map of potato chromosomes based on genomic and cDNA clones from tomato. The potato map was drawn from segregation data derived from the interspecific cross S. phureja X (S. tuberosum X S. chacoense) (2n = 2x = 24), and consists of 135 markers defining 12 distinct linkage groups. Nearly all of the tomato probes tested hybridized to potato DNA, and in most cases, the copy number of the employed clones was the same in both species. Furthermore, all clones mapped to the same linkage group in both species. For nine chromosomes, the order of loci appears to be identical in the two species, while for the other three, intrachromosomal rearrangements are apparent, all of which appear to be paracentric inversions with one breakpoint at or near the centromere. These results are consistent with cytogenetic theory, previously untested in plants, which predicts that paracentric inversions will have the least negative effect on fitness and thus be the most likely form of chromosomal rearrangements to survive through evolutionary time. Linkage maps based on a common set of restriction fragment length polymorphism markers provide a basis for uniting the previously separate disciplines of tomato and potato genetics. Using these maps, it may now be possible to test theories about homologies or orthologies of other genes, including those coding for disease resistance and stress tolerances.

Abstract A morphologically and agronomically heterogeneous collection of 38 diploid potato lines was analysed for restriction fragment length polymorphisms (RFLPs) with 168 potato probes, including random genomic and cDNA sequences as well as characterized potato genes of known function. The use of four cutter restriction enzymes and a fragment separation range from 250 to 2,000 bases on denaturing polyacrylamide gels allowed the detection of RFLPs of a few nucleotides. With this system, 90% of all probes tested showed useful polymorphism, and 95% of those were polymorphic with two or all three enzymes used. On the average, 80% of the probes were informative in all pairwise comparisons of the 38 lines with a minimum of 49% and a maximum of 95%. The percentage of heterozygosity was determined relative to each other for each line and indicated that direct segregation analysis in F1 populations should be feasible for most combinations. From a backcross involving one pair of the 38 lines, a RFLP linkage map with 141 loci was constructed, covering 690 cMorgan of the Solanum tuberosum genome.

[18]

Van Eck HJ, Van der VoortJ R, DraaistraJ, et al.

The inheritance and chromosomal localization of AFLP markers in a non-inbred potato offspring

AFLP TM is a new technique to generate large numbers of molecular markers for genetic mapping. The method involves the selective amplification of a limited number of DNA restriction fragments out of complex plant genomic DNA digests using PCR. With six primer combinations 264 segregating AFLP amplification products were identified in a diploid backcross population from non-inbred potato parents. The identity of an AFLP marker was specified by the primer combination of the amplification product and its size estimated in bases. The segregating AFLP amplification products were mapped by using a mapping population with 217 already known RFLP , isozyme and morphological trait loci. In general, the AFLP markers were randomly distributed over the genome, although a few clusters were observed. No indications were found that AFLP markers are present in other parts of the genome than those already covered by RFLP markers. Locus specificity of AFLP markers was demonstrated because equally sized amplification products segregating from both parental clones generally mapped to indistinguishable maternal and paternal map positions. Locus specificity of AFLP amplification products will allow to establish the chromosomal identity of linkage groups in future mapping studies. Since AFLP technology is a multi-locus detection system, it was not possible to identify the AFLP alleles which belong to a single AFLP locus. The consequences of a genetic analysis based on single alleles, rather than on loci with two or more alleles on mapping studies using progenies of non-inbred parents are discussed.

Based on amp lified fragment length polymorphism (AFLP) markers, a molecular linkage map of potato was initially developed by using Fsegregating population from the cross between the diploid potato parents of 08675-21 and 09901-01. The genetic map included 24 main linkage group s, and the femalemaps covered 512 cM with an average distance of 6.83 cM between adjacentmarkerswhile the male2map s covered 578 cM with an average distance of 6.08 cM between adjacent markers.

[20]

ViskerM, KeizerL, Eck HV, et al.

Can the QTL for late blight resistance on potato chromosome 5 be attributed to foliage maturity type?

We investigated the association between late blight resistance and foliage maturity type in potato by means of molecular markers. Two QTLs were detected for foliage resistance against Phytophthora infestans (on chromosomes 3 and 5) and one for foliage maturity type (on chromosome 5). The QTL for resistance to late blight and the QTL for foliage maturity type on chromosome 5 appeared to be mapped on indistinguishable positions. We were interested whether this genetic linkage was due to closely linked but different genes, or due to one (or more) gene(s) with pleiotropic effects. We therefore developed an approach to detect QTLs, in which resistance to late blight was adjusted for foliage maturity type. This analysis revealed the same two QTLs for resistance against P. infestans , but the effect of the locus on chromosome 5 was reduced to only half the original effect. This is a strong indication that the two indistinguishable QTLs for foliage maturity type and for late blight resistance on chromosome 5 may actually be one gene with a pleiotropic effect on both traits. However, there was still a significant effect on resistance against P. infestans on the locus on chromosome 5 after adjusting for foliage maturity type. Therefore we cannot rule out the presence of two closely linked QTLs on chromosome 5: one with a pleiotropic effect on both late blight resistance and foliage maturity type, and another with merely an effect on resistance. In addition, the two QTLs for resistance to late blight showed an important epistatic interaction, suggesting that QTLs for resistance affect each other's expression.

[21]

DananS, Chauvin JE, CaromelB, et al.

Major-effect QTLs for stem and foliage resistance to late blight in the wild potato relatives Solanum sparsipilum and S. spegazzinii are mapped to chromosome X

To find out new resistance sources to late blight in the wild germplasm for potato breeding, we examined the polygenic resistance of Solanum sparsipilum and S. spegazzinii by a quantitative trait locus (QTL) analysis. We performed stem and foliage tests under controlled conditions in two diploid mapping progenies. Four traits were selected for QTL detection. A total of 30 QTLs were mapped, with a large-effect QTL region on chromosome X detected in both potato relatives. The mapping of literature-derived markers highlighted colinearities with published late blight QTLs or R-genes. Results showed (a) the resistance potential of S. sparsipilum and S. spegazzinii for late blight control, and (b) the efficacy of the stem test as a complement to the foliage test to break down the complex late blight resistance into elementary components. The relationships of late blight resistance QTLs with R-genes and maturity QTLs are discussed.

Main and interaction effects of day-length and pathogen isolate on the reaction and expression of field resistance to Phytophthora infestans were analyzed in a sample of standard clones for partial resistance to potato late blight, and in the BCT mapping population derived from a backcross of Solanum berthaultii to Solanum tuberosum . Detached leaves from plants grown in field plots exposed to short- and long day-length conditions were independently inoculated with two P. infestans isolates and incubated in chambers under short- and long photoperiods, respectively. Lesion growth rate (LGR) was used for resistance assessment. Analysis of variance revealed a significant contribution of genotype isolate day-length interaction to variation in LGR indicating that field resistance of genotypes to foliar late blight under a given day-length depended on the infecting isolate. An allele segregating from S. berthaultii with opposite effects on foliar resistance to late blight under long- and short day-lengths, respectively, was identified at a quantitative trait locus (QTL) that mapped on chromosome 1. This allele was associated with positive (decreased resistance) and negative (increased resistance) additive effects on LGR, under short- and long day-length conditions, respectively. Disease progress on whole plants inoculated with the same isolate under field conditions validated the direction of its effect in short day-length regimes. The present study suggests the occurrence of an isolate-specific QTL that displays interaction with isolate behavior under contrasting environments, such as those with different day-lengths. This study highlights the importance of exposing genotypes to a highly variable population of the pathogen under contrasting environments when stability to late blight resistance is to be assessed or marker-assisted selection is attempted for the manipulation of quantitative resistance to late blight.

Using RFLP markers, QTLs for tuber starch-content and tuber yield were mapped in two F 1 populations derived from crossing non-inbred di-haploid potato breeding lines. QTLs were identified and mapped, based on both single-marker tests and interval analyses. A model specifically developed for interval QTL analysis in non-inbred plant species was successfully applied for the first time to experimental data. Results of both methods of QTL analysis were similar but not identical. QTLs for tuber starch-content and tuber yield were analysed in segregating populations K31 and LH in five and two environments, respectively. Population K31 was fully genotyped whereas population LH was selectively genotyped according to high and low tuber-starch content. Eighteen putative QTLs for tuber starch-content were identified on all 12 potato linkage groups and eight putative QTLs for tuber yield were identified on eight linkage groups. Twenty of twenty six putative QTLs were reproducibly detected in at least two environments and/or mapping populations. Few major QTLs for tuber starch-content were highly stable across environments but were detected in only one of the two mapping populations analysed. Most QTLs for tuber yield were linked with QTLs for tuber starch-content suggesting that the effects on both traits are controlled by the same genetic factors. The results are discussed with respect to marker-assisted selection in potato.

The potato P locus is required for the production of blue/purple anthocyanin pigments in any tissue of the potato plant such as tubers, flowers, or stems. We have previously reported, based on RFLP mapping in tomato, that the gene coding for the anthocyanin biosynthetic enzyme flavonoid 3′,5′-hydroxylase ( f3 ′ 5 ′ h ) maps to the same region of the tomato genome as P maps in potato. To further evaluate this association a Petunia f3 ′ 5 ′ h gene was used to screen a potato cDNA library prepared from purple-colored flowers and stems. Six positively hybridizing cDNA clones were sequenced and all appeared to be derived from a single gene that shares 85% sequence identity at the amino acid level with Petunia f3 ′ 5 ′ h . The potato gene cosegregated with purple tuber color in a diploid F 1 sub-population of 37 purple and 25 red individuals and was found to be expressed in tuber skin only in the presence of the anthocyanin regulatory locus I . A potato f3 ′ 5 ′ h cDNA clone was placed under the control of a doubled CaMV 35S promoter and introduced into the red-skinned cultivar ‘Désirée’. Tuber and stem tissues that are colored red in Désirée were purple in nine of 17 independently transformed lines.

[28]

De Jong WS, Eannetta NT, De Jong DM, et al.

Candidate gene analysis of anthocyanin pigmentation loci in the Solanaceae

Crop species in the Solanaceae , which includes tomato ( Lycopersicon esculentum ), potato ( Solanum tuberosum ), pepper ( Capsicum spp. ), and eggplant ( S. melongena ), exhibit natural variation in the types, levels, and tissue-specific expression patterns of anthocyanin pigments. While the identities of the genes underpinning natural variation in anthocyanin traits in these crops are largely unknown, many structural genes and regulators of anthocyanin biosynthesis have been isolated from the solanaceous ornamental species Petunia . To identify candidate genes that may correspond to loci controlling natural variation in the four crops, 13 anthocyanin-related genes were localized on a tomato F 2 genetic map. Gene map positions were then compared to mapped mutants in tomato and through comparative genetic maps to natural variants in potato, eggplant, and pepper. Similar map positions suggest that the tomato mutants anthocyaninless , entirely anthocyaninless , and anthocyanin gainer correspond to flavonoid 3′5′-hydroxylase ( f3′5′h ), anthocyanidin synthase, and the Petunia Myb domain trancriptional regulatory gene an2 , respectively. Similarly potato R , required for the production of red pelargonidin-based pigments, P , required for production of purple delphinidin-based pigments, and I , required for tissue-specific expression in tuber skin, appear to correspond to dihydroflavonol 4-reductase, f3′5′h and an2 , respectively. The map location of an2 also overlaps pepper A and eggplant fap 10.1, lla 10.1, lra 10.1, sa 10.1, pa 10.1 and ca 10.1, suggesting that a homologous regulatory locus has been subjected to parallel selection in the domestication of many solanaceous crops. To test the hypothesis that tomato anthocyaninless corresponds to f3′5′h , a portion of the gene was sequenced. A premature stop codon was observed in an anthocyaninless mutant, but not in wild-type.

Noncommercial varieties of potatoes ( Solanum tuberosum L.) harbor genetic potential for improvements of disease resistance and abiotic stress tolerance in commercial potato cultivars; however, introducing traits from noncommercial varieties to breeding stock can be extremely labor intensive. Molecular genetic markers closely associated with a trait can be used to decrease the time spent phenotyping varieties. Here we review genetic markers that have been used for marker-assisted selection (MAS) in potato. Most MAS markers have been used to detect disease resistance genes, and our review focuses on those markers. Complex traits such as cold, drought and viral tolerance can be studied by comparing expressed genes; next-generation sequencing technologies will help in the discovery of trait-specific molecular markers. This review aids in summarizing the potential of these molecular tools when breeding for complex traits in potato.

Phytophthora infestans remains a problem to production agriculture. Historically there have been many controversies concerning its biology and pathogenicity, some of which remain today. Advances in molecular biology and genomics promise to reveal fascinating insight into its pathogenicity and biology. However, the plasticity of its genome as revealed in population diversity and in the abundance of putative effectors means that this oomycete remains a formidable foe.

Phytophthora infestans (Mont.) de Bary is the most important fungal pathogen of the potato ( Solanum tuberosum ). The introduction of major genes for resistance from the wild species S. demissum into potato cultivars is the earliest example of breeding for resistance using wild germplasm in this crop. Eleven resistance alleles ( R genes ) are known, differing in the recognition of corresponding avirulence alleles of the fungus. The number of R loci, their positions on the genetic map and the allelic relationships between different R variants are not known, except that the R1 locus has been mapped to potato chromosome V The objective of this work was the further genetic analysis of different R alleles in potato. Tetraploid potato cultivars carrying R alleles were reduced to the diploid level by inducing haploid parthenogenetic development of 2 n female gametes. Of the 157 isolated primary dihaploids, 7 set seeds and carried the resistance alleles R1, R3 and R10 either individually or in combinations. Independent segregation of the dominant R1 and R3 alleles was demonstrated in two F 1 populations of crosses among a dihaploid clone carrying R1 plus R3 and susceptible pollinators. Distorted segregation in favour of susceptibility was found for the R3 allele in 15 of 18 F 1 populations analysed, whereas the RI allele segregated with a 1:1 ratio as expected in five F 1 populations. The mode of inheritance of the R10 allele could not be deduced as only very few F 1 hybrids bearing R10 were obtained. Linkage analysis in two F 1 populations between R1, R3 and RFLP markers of known position on the potato RFLP maps confirmed the position of the R1 locus on chromosome V and localized the second locus, R3 , to a distal position on chromdsome XI.

[36]

EI-KharbotlyA, JacobsJ M E, te HekkertB T, et al.

Localization of Ds-transposon containing T-DNA inserts in the diploid transgenic potato:linkage to the R1 resistance gene against Phytophthora infestans (Mont.) de Bary

ABSTRACT Potato(SolanumtuberosumL.)lateblight,causedbyPhytophthora infestans (Mont.) de Bary, is one of the most damaging diseases in any crop. Deployment of resistant varieties is the most effective way to control this disease. However, breeding for late blight resistance has been a challenge because the race-specific resistance genes intro- gressed from wild potato S. demissum Lindl. have been short lived and breeding for ''horizontal'' or durable resistance has achieved only moderate successes. We previously demonstrated that the high- level late blight resistance in a wild potato relative, S. bulbocastanum Dunal subsp. bulbocastanum, is mainly controlled by a single resis- tance gene RB. Transgenic potato lines containing the RB gene have showed strong late blight resistance, comparable to the backcrossed progenies derived from the somatic hybrids between potato and S. bulbocastanum. Here we report the development of a polymerase chain reaction-based DNA marker for tracking the RB gene in breed- ing populations derived from the potato 3 S. bulbocastanum somatic hybrids. Several marker-positive breeding lines showed the expected late blight resistance in greenhouse evaluations. Our results demon- strate that marker-based selection will allow us to effectively transfer the RB gene into potato using traditional breeding methods, an alter- native to deploying the RB gene through genetic transformation.

Late blight of potato, caused by Phytophthora infestans , is one of the most economically important diseases worldwide, resulting in substantial yield losses when not adequately controlled by fungicides. Late blight was a contributory factor in The Great Irish Famine, and breeding for resistance to the disease began soon after. Several disease-resistant cultivars have subsequently been obtained, and amongst them Sárpo Mira is currently one of the most effective. The aim of this work was to extend the knowledge about the genetic basis of the late blight resistance in Sárpo Mira and to identify molecular markers linked to the resistance locus which would be useful for marker-assisted selection. A tetraploid mapping population from a Sárpo Mira02×02Maris Piper cross was phenotyped for foliar late blight resistance using detached leaflet tests. A locus with strong effect on late blight resistance was mapped at the end of chromosome XI in the vicinity of the R3 locus. Sárpo Mira’s genetic map of chromosome XI contained 11 markers. Marker 45/XI exhibited the strongest linkage to the resistance locus and accounted for between 55.8 and 67.902% of variance in the mean resistance scores noted in the detached leaflet assays. This marker was used in molecular marker-facilitated gene pyramiding. Ten breeding lines containing a late blight resistance locus from cultivar Sárpo Mira and the Rpi - phu1 gene originating from the late blight resistant accession of Solanum phureja were obtained. These lines have extended the spectrum of late blight resistance compared with Sárpo Mira and it is expected that resistance in plants containing this gene pyramid will have enhanced durability.

AbstractMultiplex PCR is practically a reasonable choice for molecular marker-assisted selection in potato breeding. We had developed and were using a multiplex PCR method for selection of resistance genes to cyst nematode (), Rx1 and ). Since then, more reliable and tightly linked markers for and , and a new marker for resistance to Ry

Due to the complexity of tetrasomic inheritance, mapping studies in potato ( Solanum tuberosum L.) are generally conducted at the diploid level. In the present study we tested the feasibility of Bulked Segregant Analysis (BSA) using a tetraploid offspring for the identification of AFLP markers linked to the R2 allele, which confers race-specific resistance to Phytophthora infestans . Eleven bulk-specific AFLP markers, detected in fingerprints of 205 AFLP primer combinations, could be mapped in a linkage group encompassing the R2 locus. The efficiency of BSA at the tetraploid level, determined by the frequency of single-dose restriction fragments (SDRF), was much higher than expected on the basis of overall genetic dissimilarity between the parental clones. The fortuitous detection of AFLPs with linkage to the R2 allele is explained on the basis of specific genetic dissimilarity between cultivated potato and the chromosomal segment introgressed from S. demissum carrying the resistant R2 allele. AFLP markers common to those with linkage to R2 were visually recognized by their electrophoretic mobility in the AFLP fingerprint in a parental clone of a reference mapping population. Using these common AFLP markers we anchored the linkage group comprising the R2 allele to potato chromosome 4.

Abstract Phytophthora infestans, the causal agent of late blight, threatens potato production worldwide. An important tool in the management of the disease is the use of resistant varieties. Eleven major resistance genes have been identified and introgressed from Solanum demissum. However, new sources of resistance are continually sought. Here, we report the characterization and refined genetic localization of a resistance gene previously identified as Rber in a backcross progeny of Solanum tuberosum and Solanum berthaultii. In order to further characterize Rber, we developed a set of P. infestans isolates capable of identifying each of the 11 R-genes known to confer resistance to late blight in potato. Our results indicate that Rber is a new resistance gene, different from those recognized in S. demissum, and therefore, it has been named RPi-ber according to the current system of nomenclature. In order to add new molecular markers around RPi-ber, we used a PCR-based mapping technique, named MASP-map, which located RPi-ber in a 3.9 cM interval between markers CT240 and TG63 on potato chromosome X. The location of RPi-ber coincides with an area involved in resistance to different pathogens of potato and tomato.

[44]

WangM, AllefsS, van den BergR G, et al.

Allele mining in Solanum:conserved homologues of Rpi-blb1 are identified in Solanum stoloniferum

Abstract Allele mining facilitates the discovery of novel resistance (R) genes that can be used in breeding programs and sheds light on the evolution of R genes. Here we focus on two R genes, Rpi-blb1 and Rpi-blb2, originally derived from Solanum bulbocastanum. The Rpi-blb1 gene is part of a cluster of four paralogues and is flanked by RGA1-blb and RGA3-blb. Highly conserved RGA1-blb homologues were discovered in all the tested tuber-bearing (TB) and non-tuber-bearing (NTB) Solanum species, suggesting RGA1-blb was present before the divergence of TB and NTB Solanum species. The frequency of the RGA3-blb gene was much lower. Interestingly, highly conserved Rpi-blb1 homologues were discovered not only in S. bulbocastanum but also in Solanum stoloniferum that is part of the series Longipedicellata. Resistance assays and genetic analyses in several F1 populations derived from the relevant late blight resistant parental genotypes harbouring the conserved Rpi-blb1 homologues, indicated the presence of four dominant R genes, designated as Rpi-sto1, Rpi-plt1, Rpi-pta1 and Rpi-pta2. Furthermore, Rpi-sto1 and Rpi-plt1 resided at the same position on chromosome VIII as Rpi-blb1 in S. bulbocastanum. Segregation data also indicated that an additional unknown late blight resistance gene was present in three populations. In contrast to Rpi-blb1, no homologues of Rpi-blb2 were detected in any material examined. Hypotheses are proposed to explain the presence of conserved Rpi-blb1 homologues in S. stoloniferum. The discovery of conserved homologues of Rpi-blb1 in EBN 2 tetraploid species offers the possibility to more easily transfer the late blight resistance genes to potato varieties by classical breeding.

[45]

Rouppe van der VoortJ N A M, Janssen G JW, OvermarsH, et al.

Development of a PCR-based selection assay for root-knot nematode resistance (Rmc1) by a comparative analysis of the Solanum bulbocastanum and S. tuberosum genome

A PCR-based assay has been developed for marker assisted selection of root-knot nematode resistance ( Rmc1) in potato. To this end, a comparative genome analysis was carried out between Solanum bulbocastanum and S. tuberosum to identify PCR-based chromosome 11 alleles linked to Rmc1. The use of co-migrating AFLP markers, obtained by using primer combinations previously applied for AFLP analysis of the S. tuberosum genome, failed to align the AFLP map of the S. bulbocastanum genome with the S. tuberosum map. Apparently, the S. bulbocastanum genome is genetically too distantly related to the S. tuberosum genome for this type of analysis. Cleaved amplified polymorphic sequence (CAPS) markers were more readily applied for a comparative analysis within the region of interest. Rmc1 could be localized within a 4 cM interval between markers CT182 and M39b. It is demonstrated that the resistance spectrum of Rmc1 includes not only Meloidogyne chitwoodi and the related species M. fallax but also a genetically distinct population of M. hapla. The cost-efficiency of the CAPS markers applied for Rmc1 renders this approach as an attractive alternative for screening large segregating populations of potato for root-knot nematode resistance.

ABSTRACT The Columbia root-knot nematode (Meloidogyne chitwoodi Golden et al.) is a serious pest that reduces tuber quality of potato (Solanum tuberosum L.) in the U.S. Northwest and other parts of the world. A gene, RMc1(blb), derived from the Mexican wild species Solanum bulbocastanum Dunal, encodes resistance to this pest. An F1 mapping population with >250 individuals generated from an intraspecific cross between resistant and susceptible clones of S. bulbocastanum, SB22 and PT29, respectively, was used for marker screening and genetic linkage analysis. One amplified fragment length polymorphism marker and five sequence tagged site (STS) markers cosegregated with RMc1(blb). The five STS markers were developed from bacterial artificial chromosome (BAC) end sequences of BAC clones that were derived from another wild species, S. demissum Lindl, and contained homologs of resistance gene N against tobacco mosaic virus. These markers were tested on families that were part of the introgression of RMc1(blb) into advanced breeding lines in BC5. The utility of an efficient alternative to greenhouse and field phenotypic screening was demonstrated. The results of this study confirm that molecular markers closely linked to RMc1(blb) will assist in a selection program, reducing expense and time involved in root-knot nematode screening.

[47]

Brunt AA.

The main viruses infecting potato crops//

Loebenstein G,Berger P H,Brunt A A,et al. Virus and Virus-Like Diseases of Potatoes and Production of Seed-Potatoes, 2001: 65-67.

Sequence-characterized amplified regions (SCARs) were developed, based on nucleotide differences within resistance gene-like fragments isolated from a potato plant carrying the Ryadg gene, which confers extreme resistance to potato Y potyvirus (PVY). It originates from Solanum tuberosum subsp. andigena, and a susceptible potato plant. SCARs were tested using 103 potato breeding lines and cultivars with diverse genetic backgrounds derived from Europe, North America, and Japan. Two markers showed high accuracy for detection of the Ryadg gene. The SCAR marker RYSC3 was generated only in genotypes carrying Ryadg. The SCAR marker RYSC4 was detected in all genotypes carrying Ryadg but also in four PVY-susceptible genotypes. Neither marker was detected in genotypes carrying other Ry genes originating from different species than S. tuberosum subsp. andigena. Therefore, these SCAR markers should be powerful tools in marker-assisted selection for Ryadg in potato breeding programs, and should also be useful for cloning of the Ryadg gene.

[49]

OttomanR, HaneD, BrownC, et al.

Validation and implementation of marker-assisted selection(MAS)for PVY resistance in a tetraploid potato breeding program

The gene Ry adg from S. tuberosum ssp. andigena provides extreme resistance to PVY. This gene has been genetically mapped to chromosome XI and linked PCR-based DNA markers have been identified. Advanced tetraploid russeted potato clones developed by the U.S. Pacific Northwest Potato Breeding ( ri-State) Program with Ry adg PVY resistance were used in this study. The objective of this work was to assess the usefulness of molecular markers linked to Ry adg as a tool for selecting PVY resistance in a tetraploid potato breeding program. To achieve this, a full-sib tetraploid population segregating for Ry adg was screened with molecular markers linked to Ry adg , artificially inoculated with PVY O and evaluated in the greenhouse. A large percentage (96.4%) of the segregating lines showed coincidence between molecular markers and ELISA results at 40days after inoculation. This justifies the use of molecular markers as an alternative to artificial inoculation followed by ELISA . Segregation (resistant vs. susceptible) based on ELISA and molecular marker results in the full-sib population indicated the presence of Ry adg as a simplex in the PVY resistant parent OR00030-1. Additional full-sib populations segregating for the Ry adg gene coming from OR00030-1 and from a related clone, AOR00628-3, were evaluated under field conditions. MAS can be used as a fast and efficient tool to select for PVY resistance, reducing the number of PVY susceptible lines retained for succeeding field evaluations, and thereby increasing the odds of generating PVY resistant potato varieties.

DNA markers have a large potential to improve efficiency and precision of conventional plant breeding programmes based on marker-assisted selection (MAS). In our study, we have evaluated the predictive abilities of the SCAR marker RYSC3 and the CAPS marker GP122564 with regard to the PVY resistance genes Ryadg and Rysto, respectively, and of marker TG689 linked to H1 conferring resistance to Globodera rostochiensis and marker HC associated with high levels of G. pallida resistance. The evaluations were made in 28 cultivars and accessions and in 219 progeny genotypes descending from ten different crosses. We observed in all evaluated cultivars and accessions the expected marker patterns according to their phenotypic classification into resistant and susceptible genotypes. However, in part considerable discrepancies were observed when analysing progeny of controlled crosses involving these resistance sources, particularly with respect to H1. Based on these results, practical aspects for the efficient implementation of marker-assisted selection are discussed, which consider the genetic origin of the material, costs aspects and methodology applied.

[51]

Song YS, SchwarzfischerA.

Development of STS markers for selection of extreme resistance (Rysto) to PVY and maternal pedigree analysis of extremely resistant cultivars

Two Ry sto STS markers, YES3-3A (34102bp) and YES3-3B (28602bp), were successfully developed from the Ry sto AFLP marker E+ACC/M+CTC-365. Both STS markers identified all 38 extremely resistant potato cultivars out of 188 European potato varieties from Germany, Hungary, Poland and The Netherlands, and also identified four extremely resistant MPI breeding lines out of five tested lines, in agreement with the phenotypic data of resistance level to PVY. In parallel, three different plastid-mitochondrial configurations (W/α, W/γ, and T/β) were analyzed by the PCR markers ALC_1,3 and ALM_4,5. Thirty-eight potato varieties were classified as the W/α cytoplasm type, 51 potato varieties were identified as carrying the W/γ cytoplasm type, and 99 potato varieties analyzed had the T/β cytoplasm type. All 38 marker-selected extremely resistant potato cultivars and four extremely resistant MPI breeding lines were identified as having the mitochondrial γ type in association with male sterility. Male sterility was validated by pollen germination tests in 38 potato cultivars carrying the mitochondrial α type or γ type. The male sterility of 17 extremely resistant potato cultivars and five potato cultivars carrying the W/γ cytoplasm type was confirmed by obtaining scarcely any or only few or wrinkled pollen grains and no subsequent pollen germination. On the other hand, pollen germination in all 16 potato cultivars with the W/α cytoplasm type was observed with different efficiency due to different genotypes. The Ry sto gene of extremely resistant potato cultivars and MPI breeding lines traced back to Solanum stoloniferum , and the maternal pedigree analysis of these cultivars clustered into three groups depending on three different primary crosses with S. stoloniferum -hybrids. This pedigree analysis confirmed the precision of Ry sto STS marker selection tagging the maternally inherited Ry sto gene derived from MPI breeding lines.

[52]

FlisB, HennigJ, MarczewskiW, et al.

The Ry-fsto gene from Solanum stoloniferum for extreme resistant to potato virus Y maps to potato chromosome Ⅻ and is diagnosed by PCR marker GP122718 in PVY resistant potato cultivars

A novel locus for extreme resistance to Potato virus Y (PVY), Ry-f sto , was identified on potato chromosome XII. The gene Ry-f sto has been introgressed from the wild potato species Solanum stoloniferum . Inheritance of Ry-f sto in the tetraploid potato population Rysto was consistent with the model of a single, dominant gene. Bulked segregant analysis identified an ISSR (inter-simple sequence repeat) marker UBC 857 980 linked to Ry-f sto . This marker mapped to linkage group XII of a reference potato RFLP (restriction fragment length polymorphism) map. Chromosome XII specific RFLP markers were converted into PCR-based STS and CAPS markers and tested for linkage with Ry-f sto in the population Rysto. CAPS marker GP122 718 was tightly linked to the resistance gene and was successfully used to identify Polish and German cultivars expressing extreme resistance to PVY. This indicates that the source of Ry-f sto has been widely utilized in various potato breeding programs and can be monitored by a diagnostic marker in marker-assisted selection.

A simple and robust multiplex PCR approach was developed for detection of the alleles Ry-f sto and Ns conferring resistance of potato to Potato Virus Y (PVY) and Potato Virus S (PVS), respectively. Cleaved amplified polymorphic sequence (CAPS) markers GP122 564 linked to Ry-f sto and SC811 260 linked to Ns were amplified in one PCR reaction and identified after simultaneous digestion of the amplicons with restriction enzymes Eco RV and Mbo I. Effectiveness of this procedure for marker-assisted selection was confirmed in 55 potato cultivars.

[54]

HosakaK, HosakaY, MoriM, et al.

Detection of a simplex RAPD marker linked to resistance to potato virus Y in a tetraploid potato

Extreme resistance to potato virus Y, derived from a wild diploid species Solanum chacoense , was found in Japanese cultivar Konafubuki. The segregation ratio of resistant vs susceptible in the tetraploid population from Kita-akari (susceptible) x Konafubuki (resistant) indicated that the resistance gene followed a monogenic dominant fashion. Bulked DNA samples of resistant and of susceptible clones were screened with 306 decamer primers by PCR to find RAPD markers linked to the resistance. The RAPD marker 38-530 was reproducibly detected in the resistant clones with a recombination frequency of 16.3%. Except for Konafubuki the marker band was found only in a few limited parental lines and cultivars where the resistance is not involved. Thus, using Konafubuki as a resistance gene source, the RAPD marker 38-530 would be practically and widely useful in tetraploid breeding programs.

[55]

Celebi-ToprakF, Slack SA, Jahn MM.

A new gene,Nytbr,for hypersensitivity to potato virus Y from Solanum tuberosum maps to chromosome Ⅳ

A diploid backcross population derived from a cross between Solanum tuberosum and Solanum berthaultii segregated for monogenic dominant hypersensitivity to Potato virus Y (PVY). We propose the symbol Ny tbr for this locus because plants carrying this gene develop necrosis after inoculation with PVY and the allele originated in S. tuberosum. The gene mapped to chromosome IV between TG316 and TG208 at LOD=2.72. This location does not correspond to any other mapped resistance genes in potato.

Potato virus Y (PVY) is one of the most important viruses affecting potato ( Solanum tuberosum ) production. In this study, a novel hypersensitive response (HR) gene, Ny - 2 , conferring resistance to PVY was mapped on potato chromosome XI in cultivar Romula. In cultivars Albatros and Sekwana, the Ny - 1 gene was mapped on chromosome IX. In cv. Romula, the local lesions appeared in leaves inoculated with the PVY N-Wi isolate at 20 and 2802°C; PVY systemic infections were only occasionally observed at the higher temperature. In cvs. Albatros and Sekwana, expression of the necrotic reaction to virus infection was temperature-dependent. PVY N-Wi was localized at 2002°C; at 2802°C, the systemic, symptomless infection was observed. We developed the B11.6 1600 marker co-segregating with Ny - 2 and the S1d11 marker specific for the Ny - 1 gene. Fifty potato cultivars were tested with markers B11.6 and S1d11 and marker SC895 linked to the Ny - 1 gene in cv. Rywal. These results indicated the utility of these markers for marker-assisted selection of HR-like PVY resistance in potato breeding programs.

[57]

SzajkoK, ChrzanowskaM, WitekK, et al.

The novel gene Ny-1 on potato chromosome Ⅸ confers hypersensitive resistance to potato virus Y and is an alternative to Ry genes in potato breeding for PVY resistance

Fusarium dry rot, caused by several Fusarium species , is a major storage disease of potatoes for which there is no fungicidal control. Levels of resistance in commercial potato germplasm are inadequate. The purpose of this study was to determine the inheritance of resistance to Fusarium dry rot in a diploid hybrid Solanum phureja-Solanum stenotomum population. Three tubers from each of four half-sibs from each of 38 diploid families were inoculated with a mixture of two isolates of Fusarium sambucinum and one isolate of Fusarium solani four times in both 2003-2004 and 2004-2005. Tubers were then incubated for 40 days at 15 C and 90% relative humidity. The surface diameter and depth of infected tissue were measured in two directions, at right angles to each other. Depth of infected tissue was determined by cutting the tuber in half through the inoculation point. Mean depth and diameter of infected tissue were analyzed. There were significant differences among clones. The experiment x clone interaction was also significant. Broad-sense heritabilities and their 95% confidence intervals for resistance to Fusarium dry rot in this population were estimated as 0.63 (0.50, 0.71) and 0.81 (0.76, 0.86) in 2003-2004 and 2004-2005, respectively, for mean diameter; and as 0.68 (0.57, 0.75) and 0.81 (0.75, 0.86) in 2003-2004 and 2004-2005, respectively, for mean depth. Narrow-sense heri-tabilities for mean diameter and depth were not significantly different from zero either year. Although there is genetic variation for resistance to Fusarium dry rot in this population, these results indicate that additive genetic variance is lacking or minimal, and therefore little or no genetic gain in resistance will be realized. A few highly resistant clones could, via 4x-2x crosses, theoretically transfer much of the dominance and epistatic variance governing resistance in this population to the tetraploid level.

[59]

MarczewskiW, FlisB, SyllerJ, et al.

Two allelic or tightly linked genetic factors at the PLRV.4 locus on potato chromosome XI control resistance to potato leafroll virus accumulation

A novel locus for potato resistance to potato leafroll virus (PLRV) was characterized by inheritance studies and molecular mapping. The diploid parental clone DW 91-1187 was resistant to PLRV accumulation in both inoculated plants and their tuber progeny. The resistance to PLRV accumulation present in DW 91-1187 was not transmitted to any F 1 offspring when crossed with a PLRV susceptible clone. Instead, one half of the F 1 individuals exhibited undetectable amounts of PLRV as determined by ELISA during the primary infection assay, but accumulated PLRV in their tuber progeny plants. The other half was clearly infected both in the inoculated and tuber-born plants. The inheritance of resistance to PLRV accumulation may be explained by a model of two complementary alleles of a single gene ( PLRV.4 ) or by two complementary genes that are closely linked in repulsion phase. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers linked to the PLRV.4 locus were selected. The two complementary factors were closely linked in coupling phase to the alternative alleles UBC864 600 and UBC864 800 of DNA marker UBC864. These markers may be used for marker-assisted selection of genotypes having both factors for resistance to PLRV accumulation. The PLRV.4 locus was mapped to a central position on linkage group XI of the potato molecular map, where no resistance locus has been mapped previously.

[60]

Kuhl JC, Novy RG, Jonathan LW, et al.

Development of molecular markers closely linked to the potato leafroll virus resistance gene,Rlretb,for use in marker-assisted selection

Potato leafroll virus (PLRV) is a major pathogen of potato with worldwide impact on seed and commercial production of potato. In North America, the primary varieties grown by industry are not resistant to PLRV and require the application of insecticides to control the aphid vector of PLRV, so as to prevent PLRV infection of the crop. The Solanum etuberosum- derived dominant gene Rlretb confers resistance to potato leafroll virus and has been mapped to chromosome 4. The previous closest marker to Rlretb , C2_At1g42990, was mapped 13.6 cM from the gene in a BC3 population. The development of additional molecular markers closely linked to Rlretb can significantly facilitate its use in breeding and its continued introgression into cultivated potato. Cleaved amplified polymorphic sequence markers were developed in the region surrounding C2_At1g42990 and mapped along with Rlretb in 102 BC4 progeny. Sixteen markers were identified surrounding Rlretb, with flanking markers at 2.1 and 9.3 cM. Two markers, 1367-8a and DMB32-11, both 2.1 cM from Rlretb, are shown to be well suited for marker assisted selection. Currently, breeding clones with the presence of Rlretb have been developed that comprise market classes characterized by having tubers that are round and white, or long and russet-skinned.

Major gene inheritance of resistance to Potato leafroll virus (PLRV) was demonstrated in a parthenogenic population derived from the highly resistant tetraploid a ndigena landrace, LOP-868. This major gene or chromosome region seems to control a single mechanism for resistance to infection and virus accumulation in this source. About 149 dihaploid lines segregated in a ratio of 107 resistant to 32 susceptible, fitting the expected ratio for inheritance of a duplex gene under random chromatid segregation. A tetraploid AFLP map was constructed using as reference the ultra high density (UHD) map. All AFLP markers associated with PLRV resistance mapped to the same linkage group. Map position was confirmed by analysis of previously-mapped SSR markers. Rl adg is located on the upper arm of chromosome V , at 1 cM from its most closely linked AFLP marker, E35M48.192. This marker will be used to develop allele-specific primers or a pair of flanking PCR-based markers for their use in marker assisted selection.

ABSTRACT A resistance gene analog (RGA)-derived sequence-characterized amplified region (SCAR) marker was successfully developed based on sequence homology with disease resistance genes of an AFLP molecular marker tightly linked to the Rl adg gene of Solanum tuberosum ssp. andigena. The new marker was designated as ‘RGASC850’ (RGA-derived SCAR) based on the size of the amplified fragment. ‘RGASC850’ could be efficiently used for monitoring introgression of Rl adg against backgrounds of improved gene pools with low likelihood of identifying false positives due to recombination. This SCAR proved to be highly predictive of Rl adg -based resistance, as it did not amplify potato leafroll virus (PLRV) resistance sources other than andigena, and thus would be useful in developing cultivars with complementary sources of resistance to PLRV. In addition, a cleaved amplified polymorphic sequence (CAPS) marker based on ‘RGASC850’ was developed capable of distinguishing genotypes carrying Rl adg . This CAPS marker would be useful for screening breeding populations derived from wide crosses, and confirming presence of the Rl adg gene in those parents amplifying the ‘RGASC850’ marker.

The dominant Nb gene of potato confers strain-specific hypersensitive resistance against potato virus X (PVX). A population segregating for Nb was screened for resistance by inoculating with PVX strain CP2, which is sensitive to Nb . Through a combination of bulked segregant analysis and selective restriction fragment amplification, several amplified fragment length polymorphism (AFLP) markers linked to Nb were identified. These were cloned and converted into dominant cleaved amplified polymorphic sequence (CAPS) markers. The segregation of these markers in a Lycopersicon esculentum × L. pennellii mapping population suggested that Nb is located on chromosome 5. This was confirmed by examining resistant and susceptible potato individuals with several tomato and potato chromosome-5-specific markers. Nb maps to a region of chromosome 5 where several other resistance genes– including R1 , a resistance gene against Phytophthora infestans , Gpa , a locus that confers resistance against Globodera pallida , and Rx2 , a gene that confers extreme resistance against PVX–have previously been identified.

The dominant allele Ns confers in potato resistance to Potato virus S (PVS). To identify the chromosomal location of Ns , we mapped the Ns -linked marker SCG17 448 and the ISSR marker UBC811 600 to linkage group VIII of the RFLP map of a population that did not segregate for Ns . The map position of the Ns locus on chromosome VIII was confirmed with the detection of linkage between Ns and three RFLP markers, GP126, GP189 and CP16, known to be located in a corresponding region on potato chromosome VIII. PCR-based assays were developed for these RFLP markers. The PCR primers specific for GP126 generated polymorphic products (STS marker). In the case of markers GP189 and CP16, informative polymorphism was revealed in the Ns population after digestion with the restriction enzymes Hae III and Hin dIII, respectively. The genetic distance between Ns and the closest CP16 locus was 4.2 cM.

Two new loci for resistance to potato virus M (PVM), Gm and Rm , have been mapped in potato. The gene Gm was derived from Solanum gourlayi , whereas, Solanum megistacrolobum is the source of the gene Rm . Gm confers resistance to PVM infection after mechanical inoculation. Rm induces a hypersensitive response in potato plants. Two diploid populations segregating for Gm and Rm , bulked segregant analysis (BSA) using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR), and available potato molecular maps were instrumental for mapping the resistance loci. The novel locus Gm was mapped to a central region on potato chromosome IX. The locus Rm was placed on the short arm of chromosome XI, close to the marker loci GP250 and GP283 , where a hotspot for monogenic and polygenic resistance to diverse pathogens is located in the potato and tomato genome.

[66]

BebdahmaneA, KanyukaK, Baulcombe DC.

High-resolution genetical and physical mapping of the Rx gene for extreme resistance to potato virus X in tetraploid potato

The Rx locus in potato confers extreme resistance to PVX. In the F 1 progeny of crosses between the PVX-susceptible cultivar Huinkel and the cultivar Cara ( Rx genotype) there was a 165:651 segregation of PVX resistance, indicating that Rx in Cara is present in the simplex condition. Using potato and tomato RFLP markers, we mapped Rx in Cara to the distal end of chromosome XII at a different position to the previously mapped Rx 1 locus. To generate a high-resolution linkage map in the vicinity of Rx a total 728 AFLP primer combinations were screened using DNA of bulked resistant and susceptible segregants. We also screened segregating populations for chromosomal recombination events linked to the Rx locus and identified 82 plants with recombination events close to Rx . Using these recombinant plants we have identified AFLPs that flank Rx and span an interval of 0.23 cM in a region of the genome where 1 cM corresponds to approximately 40061kb.

The diseases considered in this chapter range from those caused by persistent soil borne fungi to those which are tuber-borne. The diseases are discussed in an order which reflects the extent to which the genetics of resistance is understood: Synchytrium endobioticum, dry rot (Fusarium solani, F. sulphureum [Gibberella cyanogena], F. sambucinum [G. pulicaris] and G. avenacea), Rhizoctonia solan...

[68]

GebhardtC, BellinD, HenselewskiH, et al.

Marker-assisted combination of major genes for pathogen resistance in potato

Abstract Closely linked PCR-based markers facilitate the tracing and combining of resistance factors that have been introgressed previously into cultivated potato from different sources. Crosses were performed to combine the Ry ( adg ) gene for extreme resistance to Potato virus Y (PVY) with the Gro1 gene for resistance to the root cyst nematode Globodera rostochiensis and the Rx1 gene for extreme resistance to Potato virus X (PVX), or with resistance to potato wart (Synchytrium endobioticum). Marker-assisted selection (MAS) using four PCR-based diagnostic assays was applied to 110 F1 hybrids resulting from four 2x by 4x cross-combinations. Thirty tetraploid plants having the appropriate marker combinations were selected and tested for presence of the corresponding resistance traits. All plants tested showed the expected resistant phenotype. Unexpectedly, the plants segregated for additional resistance to pathotypes 1, 2 and 6 of S. endobioticum, which was subsequently shown to be inherited from the PVY resistant parents of the crosses. The selected plants can be used as sources of multiple resistance traits in pedigree breeding and are available from a potato germplasm bank.

Two different chromosomal locations of major genes controlling extreme resistance to potato virus X (PVX) were found by restriction fragment length polymorphism ( RFLP ) analysis of two populations segregating for the resistance. The resistance gene Rx1 mapped to the distal end of chromosome XII, whereas Rx2 was located at an intermediate position on linkage group V in a region where reduced recombination and segregation distortion have also been observed. These linkage anomalies were due to abnormal behaviour of the chromosome contributed by the resistant parent P34. The results presented were obtained using two different strategies for mapping genes of unknown location. One approach was the use of probes revealing polymorphic loci spread throughout the genome and resulted in the mapping of Rx1 . The second approach was based on the assumption of possible linkage between the resistance gene and clone-specific DNA fragments introduced from a wild potato species. Rx2 was mapped by adopting this strategy.

The line IvP35 of the diploid (2n=2x=24) cultivated potato species Solanum phureja (family Solanaceae) expresses hypersensitive resistance (H) to potato X potexvirus (PVX). In this study, a diploid potato population was produced using IvP35 as the male parent and a diploid line of S. tuberosum (87HW13.7) as the female parent and tested for resistance to PVX. Data indicated that H to PVX in IvP35 is a dominant, monogenically inherited trait controlled by a single gene, named Nx phu , that is in a simplex condition ( Nxnx ). RFLP analysis carried out on the progeny lines revealed 4 markers (CT220, TG328, CT112 and TG424) from the long arm of chromosome IX that were linked to the hypersensitive phenotype; the closest linkage was observed with the marker TG424. Previous authors have shown that the same region of chromosome IX contains the gene Sw-5 for resistance to tomato spotted wilt tospovirus in Lycopersicon peruvianum (Solanaceae).

One hundred and eighty dihaploid clones used for protoplast fusions, and 144 tetraploid German potato cultivars were analysed for their cytoplasms using 11 homologous mt DNA-probes, and were classified as mitochondrial (mt) types α, β, γ, δ, and 07 according to their RFLP patterns. From the 4 x cultivars, 79 had the typical mt-type β of Solanum tuberosum being different from the 46 cvs which had the mt-α type and 19 others with mt-γ. A dendrogram shows their relationships to other Solanum species. The distantly related mt-07 was only found in di-haploids, and particularly in clones deriving from Solanum phureja and Solanum andigena . Accessory mt types will be actualized on website (http://www.edv.agrar.tu-muenchen.de/pbpz/ mm/mt/al1.htm). In order to evaluate the genetic potential of novel plastid-mitochondrial configurations we have analyzed four representative populations, which derive from different fusion-combination classes: [α (+) β], [α (+) γ], [α (+) δ] and [α (+) 07]. On the mitochondrial expression level, hybrids from an [α (+) 07] fusion could be distinguished by in-organello translation from [α (+) β] hybrids, and other di-haploids, by an additional translation product of 1561kDa. In fusion parents with mt-α and -γ an additional atp6 reading frame is detectable in sub-stoichiometric amounts by the use of specific PCR primers. The gene differs from the original 211 bp 3′ from the stop codon. Novel RFLP -patterns in 10% of the somatic hybrids were due to a high-rate replication of this pre-existing parental genome region. A second characteristic for somatic hybrids was the partial addition of parental mt sub-genomes. The major part of them revealed a new organization in their mt genomes at the mt-type characteristic loci rpl5 , rps14 , cob, rps10 , coxI and rpl2 , which contain recombination-specific repeats homologous to Petunia spp. and Nicotiana . A schematic model for the formation of novel mitochondrial genomes in potato somatic hybrids is provided.

A Mexican hexaploid wild potato species, Solanum demissum (dms), was only used as a female in previous breeding programs. The resulting clones with dms cytoplasm produced abundant, but non-functional pollen. A 170 bp DNA fragment, named Band 1, was originally detected in the F(1) hybrid between dms and S. tuberosum. In this study, the sequenced region was extended to 1,032 bp; nevertheless, it did not show any homology to known sequences. This extended region harboring Band 1 was, without introns, all transcribed to mRNA and was maternally inherited from dms to S. tuberosum through backcrosses. Three dms accessions, 168 accessions of 38 cultivated and closely related wild species, and 158 varieties and breeding lines were surveyed, which demonstrated that Band 1 was specific to dms and varieties and breeding lines with dms cytoplasm. Thus, Band 1 is a useful marker to distinguish dms cytoplasm, which enables us to design efficient mating combinations in breeding programs.

[73]

PowellW, BairdE, DuncanN, et al.

Chloroplast DNA variability in old and recently introduced potato cultivars

Chloroplast DNA (cpDNA) variability has been examined in a range of tetraploid European potato cultivars. The potato genotypes studied included primitive cultivars such as Lumpers (1806), Yam (1836), Myatts Ashleaf (1847) and more recently bred cultivars such as Brodick (1990). Three cpDNA pheno-types were detected and these probably represent original introductions from South America into Europe. The most common cp phenotype was the T type cpDNA (Hosaka & Hanneman, 1988) characteristic of cultivars descended from cv. Rough Purple Chili. The presence of the T type cpDNA in the cultivar Yam indicates that this genotype which is of Andigena origin shares a common cytoplasm with other 5. tuberosum ssp. tuberosum clones which have a Chilean type cytoplasm. The implications of these results are discussed in relation to the origin of the T type cpDNA. Methods for increasing the cytoplasmic diversity of the cultivated potato gene pool are proposed.

[74]

HosakaK, SanetomoR.

Development of a rapid identification method for potato cytoplasm and its use for evaluating Japanese collections

The cytoplasm of potatoes, characterized by the presence of T-type chloroplast DNA and β-type mitochondrial DNA, is sensitive to nuclear chromosomal genes that contribute to various types of male sterility. Past breeding efforts with various potato varieties have resulted in several different cytoplasms other than T/β. Varieties with Solanum stoloniferum -derived cytoplasm (W/γ) show complete male sterility, while those with S. demissum -derived cytoplasm (W/α) produce abundant, but non-functional pollen. Thus, identification of cytoplasmic types is important for designing efficient mating combinations. To date, only T-type chloroplast DNA can be accurately identified by a PCR marker. Here, we report a rapid identification technique by multiplex PCR, followed by restriction digestion with Bam HI in one reaction tube, and propose a new nomenclature for potato cytoplasm types (T, D, P, A, M, and W). Using this new technique, our collections of 748 genotypes, including 84 Japanese named varieties, 378 breeding lines and 26 landraces, and 260 foreign varieties and breeding lines, were grouped into cytoplasm types: T (73.902%), D (17.402%), P (4.502%), A (1.502%), M (0.302%), and W (2.402%). The utility of this marker system for breeding is discussed.

[75]

ProvanJ, PowellW, DewarH, et al.

An extreme cytoplasmic bottleneck in the modern European cultivated potato (Solanum tuberosum) is not reflected in decreased levels of nuclear diversity. Proceedings

We have used the polymorphic chloroplast (cp) and nuclear simple sequence repeats (SSRs) to analyse levels of cytoplasmic and nuclear diversity in the gene pool of the European cultivated potato (Solanum tuberosum sap. tuberosum). Primers designed from the complete chloroplast sequence of tobacco (Nicotiana tabacum) were used to amplify polymorphic products in a range of potato cultivars. Combining the data from seven polymorphic cpSSR loci gave 26 haplotypes, one of which (haplotype A) accounted for 151 out of the 178 individuals studied and corresponded to the T-type cytoplasm previously identified in cultivated potatoes using chloroplast restriction fragment length polymorphism analysis. Phylogenetic and diversity analyses of the relationships between cpSSR haplotypes confirmed much higher levels of cytoplasmic diversity outwith the T-type group. Diversity levels at eight nuclear SSR loci, however, were not significantly different between cytoplasmic groups, suggesting a severe maternal bottleneck in the evolution of the modern cultivated potato. These results highlight the importance in quantifying levels of cytoplasmic as well as nuclear diversity and confirm the need for a change in breeding practices to increase levels of non-T-type cytoplasm in the cultivated gene pool, thus helping reduce problems associated with pollen sterility. This may be facilitated by germplasm analysis using cpSSRs, which will allow efficient selection of diverse cytoplasm donors.